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Characterisation of loci that encode immunoprotective antigens of Eimeria maxima identified through genetic linkage analyses - OD0551

Description
The protozoan Eimeria species are ubiquitous parasites that infect all livestock in a host-specific manner (i.e. the seven species that infect the chicken do not parasitise other hosts). Many species of livestock are exposed to eimerian infection, most notably intensively-reared poultry such as chickens and turkeys. In the absence of effective control, infection can result in the disease coccidiosis, characterised by lethargy, poor performance and mortality rates as high as 50%. Current control is based primarily on the use of medication (more than 230 tonnes of coccidiostatic compounds have been sold in the UK every year since 2000), but this has been compromised by the rise of drug resistance and concerns about food chain contamination. As a direct consequence legislation is becoming increasingly restrictive, dramatically reducing the number of chemicals available. The leading alternative control strategy is vaccination by exposure to infection with live parasites. Unfortunately, the costs associated with producing complex live parasite vaccines, comprising as many as eight parasite lines, has mostly limited the use of vaccination to the breeder and egglaying
sectors even though 98.1% of the estimated cost of coccidiosis has been attributed to the broiler sector. An effective, economically viable means of controlling the Eimeria species is required to improve poultry production and
welfare.

Natural infection with parasites of the Eimeria species induces a strong protective immune response in the host. Access to the components of the parasite that stimulate protective immunity will provide a rational basis for the development of a subunit or recombinant vaccine that is protective. However, whilst numerous trials with many proteins have been undertaken not one has offered the prospect of success and the key protective antigens have yet to be discovered. The identification of these antigens has previously been hampered by our inability to differentiate antigens that stimulate an immune response but provide no protection from those that stimulate a protective immune response.

A classical genetic mapping strategy that utilises two distinct strains of Eimeria maxima as the parents of a hybrid population has been developed to identify regions of the parasite genome eliminated by strain-specific immune
selection. In this manner we have focused the search for genes that encode protective antigens on just three regions of the E. maxima genome, predicted to represent approximately 80 genes from a total pool of 6,000-8,000. In the
forthcoming phases of this strategy we will sequence the identified regions of the E. maxima genome and predict the genes that they represent. The candidate genes most likely to encode protective antigens, selected based upon criteria including polymorphism between the parental strains and ability to stimulate immune mechanisms known to be stimulated in the host by eimerian infection will be used in vaccination trials.
Objective
The overall objective of the work described here is to identify those genes within the Eimeria maxima genome that encode antigens which confer protection during natural infection. Our development of a novel population-based strategy to map loci integral to strain-specific immunity in the E. maxima genome has resulted in the identification of three regions of ~100-230 Kb under immune selection, represented by a panel of bacterial artificial chromosome (BAC) clones. The remaining objectives for this project divide in to a series of four sequential phases.

1. BAC clone sequencing and identification of BAC regions under immune selection.
a. Sequence BAC clones previously shown to represent regions of the E. maxima W strain genome lost from a hybrid population under W strain-specific immune selection.
b. Define the range of each sequenced region under immune selection by (a) predicting AFLP markers from each BAC sequence and (b) examining existing AFLP data to compare the inheritance of each predicted marker by hybrid parasite populations before and after immune selection. Production of a multiple backcrossed, immune-selected, recombinant parasite population for AFLP characterisation will enhance the linkage between key DNA markers and loci under immune selection. The exclusion of BAC-encoded E. maxima sequence unaffected by immune selection will reduce the quantity of sequence to be investigated.

2. Bioinformatic identification of genes associated with immune protection and selection of genuine vaccine candidates.
a. Predict genes encoded within the sequenced regions utilising annotation software developed for the Eimeria tenella genome project, expressed sequence data from three Eimeria species including E. maxima and database homology searches.
b. Identify those predicted genes that are polymorphic at the amino acid level between the H and W strains of E. maxima (the parents of the cross used for the genetic mapping phases) through comparison with existing H strain expressed sequence data, supplemented by targeted H strain DNA and/or cDNA sequencing.

3. Determination of the immunogenicity and protective capacity of selected parasite antigens.
a. Small scale antigen expression for use in assays including T cell proliferation.
b. Candidate antigens will be tested for ability to stimulate T cells from infected (and uninfected) birds ex vivo in T cell proliferation assays and by real-time RT-PCR for chicken interferon-?.
c. Immunisation trials using a DNA prime/DNA boost or DNA prime/recombinant antigen boost approach with up to ten of the most promising candidates.

The culmination of this body of work is likely to yield one, or more, protective antigens suitable for inclusion in vaccination strategies effective against E. maxima. Homology-led searches for related antigens in Eimeria acervulina and E. tenella, using expressed and genomic sequence databases, will provide the basis for further vaccine trials. Combined, these three species are the most economically important eimerian parasites that infect the chicken.
Time-Scale and Cost
From: 2006

To: 2009

Cost: £115,986
Contractor / Funded Organisations
BBSRC Central Office
Keywords
Animal Health              
Avian Coccidiosis              
Avian Diseases              
Plants and Animals              
Fields of Study
Animal Health